Alternative pre-mRNA splicing contributes significantly to genetic diversity in vertebrates. This proposal is aimed at understanding the mechanisms underlying the regulation of alternative pre-mRNA splicing. Alternatively spliced regions of a pre-mRNA are characterized by the presence of splicing enhancer or silencer sequence elements recognized by splicing factors. Our work concentrates on four systems of pre-mRNA splicing that involve different types of regulatory elements and splicing factors. Included in the RNA regulatory sequence elements to be studied are: 1) C/A-rich exon enhancers that stimulate exon inclusion, G/A-rich exon enhancers that stimulate exon inclusion and regulate 5' splice site usage, G-rich intron enhancers that regulate 5' splice site usage, and C-rich intron elements that stimulate splicing and spliceosome assembly. Experiments are described to: 1) continue characterization of the factors that recognize these RNA elements, 2) identify and characterize factors that recognize element-binding factors to regulate splicing, 3) determine the role of RNA helicases in alternative splicing and 4) investigate the hypothesis that alternative splicing and transcriptional elongation are linked.